A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin

Background Bivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes. Results Here, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics. Conclusions Our optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-024-00527-9.

Note: all steps must be performed on ice or at 4 degrees unless otherwise specified.
Note: chromaFn fixaFon can also be performed on cells in suspension by resuspended a pellet of a known number of washed live cells directly into the 1% formaldehyde soluFon and incubaFng for 8 minutes on a rocker, quenching with glycine as below and then mild centrifugaFon to get a fixed cell pellet( rather than scraping cells).
1. Grow cells un6l they are 70-80% confluent across several plates.One will be used for coun6ng and the remaining for fixa6on (collec6on plates).
2. Collect and count cells on the coun6ng plate: Remove media from the coun6ng plate and wash once with DPBS.
3. Add 1ml of Trypsin or appropriate dissocia6on reagent per 10cm plate and incubate un6l cells lig off plate as single cells.
5. Take aliquot and count cells to determine total cell number per plate and therefore the total number of cells overall.37. Add 20µl of pre-washed protein A Dynabeads from step 21 to the chroma6n and incubate for at least 3 hours at 4 degrees on rotator to pre-clear the chroma6n.This is cri6cal to reduce non-specific binding and decrease background signal.

Overnight incuba5on with primary an5body -30 min and overnight
38.Take all bead-an6body tubes from the 4-degree rotator.
39. Back at the bench place the pre-cleared chroma6n sample and 1x IgG, 1 x H3K4me3 and 1 x H3K27me3 bead-an6body complexes per sample on the magnet rack un6l the solu6on clears.We recommend sikng the magne6c rack on ice to keep cool during these steps.Alterna6vely, these steps can be performed in a cold room.
40.The remaining bead-an6body mixes can be stored at 4 degrees un6l needed on day 2.
41. Take 10µl of pre-cleared chroma6n supernatant to a separate tube and label as 5% input control.Keep at 4 degrees un6l day 3.
42. Remove the supernatant from the an6body-bead complexes on the magne6c rack and discard.
43. Add 200µl of the pre-cleared chroma6n supernatant to each an6body/bead mixture.
44. e.While tubes are on the rack wash the beads with 400µl of freshly prepared 80% ethanol in molecular grade water f.Repeat step e for a total of 2 washes.
g.While the tubes are on the magnet, allow beads to air dry for up to 5 minutes.
Note -it is important not to over dry here.Proceed to DNA elu6on before the beads start to crack.h.To elute DNA, remove tubes from the magne6c rack and resuspend beads in Xµl (see below) of 10mM Tris-HCL pH8.0.
i. Incubate at room temperature for 5 minutes.c.Use remaining eluate (17µl) to prepare libraries using NEBNext ® Ultra™ II DNA Library Prep Kit or similar following manufacturer's instruc6ons.
j. Place tubes back on the magne6c rack for 5 minutes and transfer DNA solu6on to new tube.75.For downstream qPCR analysis elute in 60-80µl 10mM Tris-HCL pH8.0 and use 1µl per qPCR reac6on.76.For downstream NGS elute in 20µl 10mM Tris-HCL pH8.0.a. Use 1µl of eluate to determine the DNA concentra6on using a Qubit fluorometer.ChIP-reChIP protocol for mapping bivalent chroma6n -Eckersley-Maslin lab -2023 Version 1 Page 12 of 12 b.Use 2µl for qPCR analyses of posi6ve and nega6ve control regions (dilute 4x to give final volume of 8µl and use 1µl per reac6on).

Chroma5n fragmenta5on and primary an5body incuba5on -Day 1 Before
you start: pre-cool centrifuge; add Roche cOmplete EDTA-free protease inhibitor tablets to ChIP buffer and NP buffer and keep on ice unFl needed.Note: Unless otherwise specified all procedures should be carried out between 2-8 degrees Note: Add protease inhibitor cocktail to sufficient volumes of NP buffer and ChIP buffer.Note: NP buffer composiFon and cell lysis condiFons may need to be opFmised depending on your cell type.Note: volumes below are to process one sample corresponding to one vial of 2x10^7 cells and will result in input, in-line H3K4me3, in-line H3K27me3, IgG-IgG reChIP and bivalent K4-K27 and K27-K4 reChIP samples that can be further processed by qPCR and/or library preparaFon for sequencing.If you have more than one sample (e.g.biological replicate or other condiFon), scale volumes accordingly.We typically do not process more than 4 samples at any Fme.Note: sonicaFon can be used instead of MNase digesFon.We rouFnely use both fragmentaFon methods in performing reChIP experiments with similar results.

Chroma5n prepara5on -1 hour prepara5on 5me plus at least 3 hours incuba5on
Note: This amount of MNase and digesFon Fme will need to be Ftrated for each cell line.These

First elu5on, buffer exchange and secondary an5body incuba5on -Day 2
Add the IgG chroma6n sample to the IgG-bead complexes.§ Add the H3K4me3 chroma6n to the H3K27me3-bead complexes.§ Add the H3K27me3 chroma6n to the H3K4me3-bead complexes.
Top up each chroma6n/an6body/bead mixture with 300µl of ChIP buffer to final volume of 500µl.b.Carefully remove supernatant while on magnet making sure you do not disturb the beads.Make sure you do not let the beads dry out.c.Resuspend beads in 500µl of low salt buffer.d.Repeat steps a-c for a total of 3x low salt buffer washes, 3x high salt buffer washes, 2x LiCl buffer washes and 2x 1xTE washes.d.Place tubes on magne6c rack for 5 mins and remove supernatant.